ES2755091T3 - Fibrosis treatment procedures - Google Patents

Abstract

The 2-methoxy-3- (4'-aminophenyl) propionic acid compound (compound 34) represented by the formula: ** Formula ** or a pharmaceutically acceptable salt or stereoisomer thereof, for use in the treatment of fibrosis , in which fibrosis is selected from intestinal fibrosis or idiopathic pulmonary fibrosis.

Description

DESCRIPTION

Fibrosis treatment procedures

Background

Fibrosis is the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive procedure, for example healing, usually due to long-term injury or inflammation. Fibrosis causes affected tissues to harden and / or swell and reduces the flow of fluids through these tissues. As a result, tissues with fibrosis may not function properly.

For example, liver fibrosis can be described as the wound healing response to chronic liver injury as a result of, for example, alcohol and / or drug abuse, viral and parasitic infection (for example, hepatitis, such as hepatitis B or C), non-alcoholic steatohepatitis (ENA), iron and copper overload, and autoimmune diseases. All chronic liver diseases can lead to liver fibrosis, with the principle of causing chronic viral hepatitis B and alcoholic liver disease. The hepatic endocannabinoid system participates in pro and antifibrogenic effects by activating different signaling pathways that differentially affect the proliferation and death of fibrogenic cell types. Over time, this procedure can lead to liver cirrhosis in which the architectural organization of the functional units of the liver is disrupted so much that blood flow through the liver and liver function are disrupted.

Liver fibrosis represents a common and difficult challenge of global importance. Currently, the only curative treatment for terminal cirrhosis is transplantation, but even in the developed world, the number of donor organs available and the clinical status of the potential recipient limit the applicability of this technique. The development of fibrosis, and particularly cirrhosis, is associated with significant morbidity and mortality. Thus, there is a considerable imperative to develop antifibrotic strategies that are applicable to liver fibrosis.

Renal fibrosis causes significant morbidity and mortality as the primary acquired injury leading to the need for dialysis or kidney transplantation. Fibrosis can occur in the filtration or reabsorption component of the nephron, the functional unit of the kidney. Fibrosis can also occur in the heart, for example, cardiac fibrosis can occur as a thickening of a heart valve.

Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD) that can become symptomatic and may require surgical intervention if stricture formation occurs. Most of the traditional and novel mechanisms underlying intestinal fibrosis are associated with chronic inflammation.

Johnson et al. (Inflamm Bowel Dis, 2012, 18, 460-471), describe a series of experiments in which the antibiotic levofloxacin is used to treat intestinal inflammation in a mouse model of intestinal fibrosis that is initiated by Salmonella typhimurium infection . The results described there indicate that the eradication of the inflammatory stimulus did not prevent fibrosis.

WO20081004557 describes PPAPy agonists for induction of the expression of cationic antimicrobial peptides as immunoprotective stimulants.

WO2007010514 describes compounds that are specific for PPAR receptors and EGF receptors and the use of such compounds in the treatment of cancer.

WO2010091892 describes alkylamido compounds that can modulate the activity of PPAR and / or EGF receptors.

Yu et al., (Int. J. Biochem. Cell Biol., 2010, 42, 948-957) describe a series of experiments analyzing the potential for the treatment of steatohepatitis and fibrosis induced by a diet deficient in methionine choline (MCD) by transducing an adenovirus that expresses PPAPy.

Summary

In one aspect, the present invention provides 2-methoxy-3- (4-aminophenyl) propionic acid (compound 34) represented by the formula:

or a stereoisomer or pharmaceutically acceptable salt thereof, for use in the treatment of fibrosis, wherein the fibrosis is selected from intestinal fibrosis or idiopathic pulmonary fibrosis, as set forth in the claims.

In another aspect, the present invention provides a pharmaceutical preparation comprising a therapeutically effective amount of 2-methoxy-3- (4'-aminophenyl) propionic acid (compound 34), or a pharmaceutically acceptable salt or stereoisomer thereof, and one or more pharmaceutically acceptable carriers, for use in treating a patient suffering from fibrosis, wherein fibrosis is selected from intestinal fibrosis or idiopathic pulmonary fibrosis, as set out in the claims.

Description

Disclosed herein are, for example, methods of treating fibrosis comprising administering compounds disclosed herein to a patient. Compounds for use in such procedures are also disclosed. For example, a method of preventing or treating liver fibrosis and / or intestinal fibrosis is disclosed in a subject or patient in need or suffering from it, comprising administering a therapeutically and / or pharmaceutically acceptable amount of a compound represented by:

in which

R ¹⁰ is selected from the group consisting of H and C ¹ -C ⁶ alkyl;

R ¹¹ is selected from the group consisting of H, C ¹ -C ⁶ -alkyl and -C (O) -C ¹ -C ⁶ -alkyl;

R ⁵ is C ¹ -C ⁶ alkyl; and its pharmaceutically acceptable salts.

R ⁵ can be H.

The C ¹ -C ⁶ alkyl functionalities of R ¹⁰ , R ¹¹ and / or R ⁵ can be independently selected from the group consisting of: methyl, ethyl, propyl, pentyl and hexyl. The alkyl functionality can be linear or branched. Methyl and ethyl alkyl functionalities are particularly preferred, with methyl being the most preferred alkyl functionality.

Suitably, the nitrogen can be attached to the ring at the 2 ', 3', 4 'or 5' positions. Preferably, the nitrogen binds at the 3 'or 4' position. The 4 'position is most preferred.

Compounds contemplated by way of example in the disclosure may include formulas A 'and A ", wherein R ¹⁰ , R ¹¹ and R ⁵ have been provided above and herein:

R ⁵ can be H.

The C ¹ -C ⁶ alkyl functionalities of R ¹⁰ , R ¹¹ and / or R ⁵ can be independently selected from the group consisting of: methyl, ethyl, propyl, pentyl and hexyl. The alkyl functionality can be linear or branched. Methyl and ethyl alkyl functionalities are particularly preferred, with methyl being the most preferred alkyl functionality. R ⁵ can be methyl, ethyl, propyl, or is, for example, methyl.

Also disclosed herein is a method of preventing or treating liver or intestinal fibrosis, comprising administering to a patient in need, a pharmaceutical preparation comprising an agent. PPAPy such as a compound disclosed herein. Procedures are disclosed herein to prevent or treat renal fibrosis, cardiac fibrosis, endomyocardial fibrosis, idiopathic pulmonary fibrosis, myelofibrosis, retroperitoneal fibrosis, and / or nephrogenic systemic fibrosis, which comprises administering to a patient in need, a pharmaceutical preparation that It comprises a PPAPy agent such as a compound disclosed herein. For example, the procedures disclosed herein may include procedures in which the patient is a human. Accordingly, the compounds described herein can be used in the treatment and / or prevention of these diseases / conditions.

These and other aspects and advantages of the disclosure will become apparent when considering the following figures, detailed description and claims.

Brief description of the drawings

Figure 1 represents the evolution of the body weight of the mice after the administration of CCU, JWH-133 and compound 34.

Figure 2 (A) shows an explanation of metavir scores and (B) shows the effect of CCU, JWH-133 + CCl ⁴ and compound 34+ CCU on the liver and specifies a metavir score for each group.

Figure 3 (A) represents the metavir score and (B) the metavir score adapted from livers of mice subjected to CCU, JW H -133+ CCU and compound 34+ c Cu.

Figure 4 represents the effect of c Cu, JWH-133 + CCU and compound 34+ CCU on a-SMA and collagen 1.

Figure 5 represents the study schedule for intestinal fibrosis.

Figure 6 depicts the effect of compound 34 on the colon of mice having intestinal fibrosis.

Figure 7 depicts the anti-inflammatory effect of compound 34 on the colon of mice having intestinal fibrosis.

Figure 8 depicts the effect of compound 34 on the colon of mice having intestinal fibrosis.

Figure 9 depicts the effect of compound 34 on the colon of mice having intestinal fibrosis using an immunohistochemical analysis of the main fibrosis markers (Collagen I-III, CTGF, SMAD 2/3, PDGF, a SMA and TGF-P1) .

Figure 10 depicts the effect of compound 34 on the colon of mice having intestinal fibrosis using Western blot analysis.

Figure 11 shows a body weight curve in mice treated with DSS and DSS GED.

Figure 12 shows the level of fibrosis in the colon of mice treated with DSS and DSS GED compared to control mice receiving regular water.

Figure 13 depicts the microscopic findings of the chronic colitis colon from DSS and DSS GED mice. Colon tissues were fixed in 4% PFA and cross sections (4 pm) stained with May-Grunwald-Giemsa (H&E) and Masson's trichrome. DSS mice showed a severe inflammatory degree and fibrosis in both the submucosa and the serosa compared to controls, while GED treatment determined the restoration of the normal architecture of the intestinal wall. Data are expressed as the mean ± SEM; * = p <0.05; ** = p <0.01 and *** = p <0.005 vs H ² O.

Figure 14 shows the immunohistochemistry and immunoblot of the main fibrosis markers. The photomicrograph shows a significant reduction of collagen I-III (a), a-SMA (b) in DSS mice treated with GED, compared to mice with chronic DSS-induced colitis. Protein levels of these markers were also measured by Western blot confirming the GED-induced expression profile. Representative transfer points are shown. * = p <0.05, ** = p <0.01. N = 6 mice for the control group, 8 mice for the DSS group, 8 mice for the DSS GED group.

Figure 15 depicts a photomicrograph showing significant reduction of TGF-p1 (a), SMAD 2/3 (b), CTGF (c) in DSS mice treated with GED, compared to mice with chronic DSS-induced colitis. .

Figure 16 shows the effect of GED administration on IL-13 protein levels in mice treated with chronic DSS and its downstream mediators, TGF-p1 and CTGF. IL-13, TGF-p1 and CTGF protein levels were determined by Western blot analysis. Data are presented as a percentage of the mean values of the controls. Representative transfer points are shown. ** = p <0.01..N = 6 mice for the control group, 8 mice for the DSS group, 8 mice for the DSS GED group.

Detailed description

The disclosure is based, in part, upon discovering that certain compounds disclosed herein have the ability to prevent or treat fibrosis, eg, in the liver or colon. In one aspect, the disclosure relates to procedures for preventing or treating fibrosis, eg, liver fibrosis and / or intestinal fibrosis, in patients in need. The disclosed methods comprise administering a compound disclosed herein.

Before a more detailed description, certain terms used in the attached specification, examples and claims are set forth herein. These definitions should be read in light of the rest of the disclosure and should be understood by a person skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning that one skilled in the art commonly understands.

A "patient", "subject" or "host" to be treated by the subject procedure can mean a human or non-human animal, for example, a small mammal, such as a mouse or a rat, and including horse, cow, dog, cat etc.

The term "therapeutic agent" is recognized in the art and refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally and / or systemically on a subject. Examples of therapeutic agents, also known as "drugs," are described in well-known literature references, such as the Merck index, the Physicians Desk Reference, and the Pharmacological Basis of Therapeutics, and include, but are not limited to, medications; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of an illness or disease; substances that affect the structure or function of the body; or prodrugs, which become biologically active or more active after being placed in a physiological environment.

The term "therapeutic effect" is recognized in the art and refers to a local and / or systemic effect in animals, particularly mammals, and, more particularly, humans caused by a pharmacologically active substance. Therefore, the term means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of diseases or in the improvement of the development and / or desirable physical or mental conditions in an animal or human being. The term "therapeutically effective amount" means the amount of said substance that produces some desired local or systemic effect in a reasonable benefit / risk ratio applicable to any treatment. The therapeutically effective amount of said substance will vary depending on the subject and the condition of the disease to be treated, on the weight and age of the subject, the severity of the disease or condition, the form of administration and the like, that a person skilled in the art can easily determine. For example, certain compositions of the present disclosure may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

The term "treat" is recognized in the art and refers to curing as well as improving at least one symptom of any condition or disease.

The term "alkyl" is recognized in the art and includes saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain aspects of the disclosure, a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (eg, C ¹ -C ³⁰ for straight chain, C ³ -C ³⁰ for branched chain) and alternatively about 20 or less, for example from 1 to 6 carbons. Likewise, cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively, about 5, 6 or 7 carbons in the ring structure. The term "alkyl" is also defined to include halogen-substituted alkyl.

On the other hand, the term "alkyl" (or "lower alkyl") includes "substituted alkyls", which refers to alkyl moieties having substituents that replace a hydrogen on one or more carbons of the main hydrocarbon chain. Such substituents may include, for example, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thiophormate), an alkoxy, a phosphoryl, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Those skilled in the art will understand that substituted residues on the hydrocarbon chain may themselves be substituted, if appropriate. For example, substituents on a substituted alkyl can include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl, and sulfonate), and silyl groups, as well such as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CN, and the like. Examples of substituted alkyls are described below. The cycloalkyls may further be substituted with alkyls, alkenyls, alkoxies, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CN, and the like.

The terms ortho, meta and para are recognized in the art and refer to 1,2, 1,3- and 1,4-disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and ortho-dimethylbenzene are synonyms.

The definition of each expression, eg, alkyl, m, n, and the like, when occurring more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

Certain compounds contained in the compositions of the present disclosure may exist in particular geometric or stereoisomeric forms. Furthermore, the compounds of the present disclosure can also be optically active. The present disclosure contemplates all of these compounds, including cis and trans isomers , R and S enantiomers, diastereomers, (D) -isomers, (L) -isomers, racemic mixtures thereof, and other mixtures thereof, found within of the scope of disclosure. In a substituent, such as an alkyl group, there may be additional asymmetric carbon atoms. All such isomers, as well as mixtures thereof, are intended to be included in this disclosure document.

It will be understood that "substitution" or "substituted with" includes the implicit condition that such substitution agrees with the allowed valence of the substituted atom and the substituent and that the substitution results in a compound stable, for example, that does not transform spontaneously, such as by reorganization, cyclization, elimination, or other reaction.

The term "substituted" is contemplated to include all permitted substituents of organic compounds. In a broad aspect, permitted substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Substituents Illustrative include, for example, those described herein above. Allowed substituents may be one or more and the same or different suitable organic compounds. For the purposes of the present disclosure, heteroatoms, such as nitrogen, may have substituents of Hydrogen and / or any allowable substituents of organic compounds described herein that satisfy the valencies of the heteroatoms The present disclosure is not intended to be limited in any way by the permitted substituents of organic compounds.

For the purposes of the present disclosure, the chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside. Also for the purposes of this disclosure, the term "hydrocarbon" is contemplated to include all allowable compounds having at least one hydrogen atom and one carbon atom. In a broad aspect, permitted hydrocarbons include branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic acyclic and cyclic organic compounds that may be substituted or unsubstituted.

The term "pharmaceutically acceptable salts" is recognized in the art and refers to the relatively non-toxic, inorganic and organic acid addition compound salts, including, for example, those contained in the compositions of the present disclosure.

The term "pharmaceutically acceptable carrier" is recognized in the art and refers to a pharmaceutically acceptable carrier or composition material, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting any composition of the subject or component thereof from an organ or portion of the body, to another organ or part of the body. Each vehicle must be "acceptable" in the sense of being compatible with the composition of the subject and its components and not be harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other compatible non-toxic substances used in pharmaceutical formulations.

Compounds

Compounds contemplated for use in one or more of the disclosed processes include compounds represented by formula I, or a pharmaceutically acceptable salt, enantiomer, or stereoisomer thereof:

in which:

R1 and R2 are each independently selected from the group consisting of H and C ¹ alkyl - ^6; or R1 and R2 can be taken together with the nitrogen atom to which they are attached to form an aromatic or aliphatic ring with 5 or 6 atoms; Y and Z are independently selected from the group consisting of H, OH, COOH, -OR3, -CH (ORa) COOH; and

R3 is selected from the group consisting of H, phenyl, benzyl, vinyl, allyl, C ^1-6 alkyl, or C ^1-6 alkyl substituted with one or more halogens; or a pharmaceutically acceptable salt thereof.

Suitably, C ¹ -C ⁶ alkyl functionalities can be independently selected from the group consisting of: methyl, ethyl, propyl, pentyl and hexyl. The alkyl functionality can be linear or branched. Methyl and ethyl alkyl functionalities are particularly preferred, with methyl being the most preferred alkyl functionality.

And it can be H or COOH. For example, Y can be H and Z can be CH (OR3) COOH or Y can be COOH and Z can be -OR3. R3 can be methyl, ethyl, n-propyl, or isopropyl.

The NR ¹ R ² moiety of formula I may be in the 4 'position or it may be in the 3' position. For example, R ¹ and R ² can be H.

Suitably R ³ can be H.

The compounds disclosed herein also include those represented by formulas IIa or IIb or a pharmaceutically acceptable salt, enantiomer, or stereoisomer of:

in which:

R1 and R2 are independently selected from the group consisting of H and C ¹ alkyl - ^6; or R1 and R2 can be taken together with the nitrogen atom to which they are attached to form an aromatic or aliphatic ring with 5 or 6 atoms; R6 is selected from the group consisting of -NR9OH, OH and -OR9;

R9 is alkyl C ^1-6;

R4 is selected from the group consisting of H, halo, phenyl, benzyl, vinyl, allyl, C ^1-6 alkyl, and C ^1-6 alkyl substituted with one or more halogens;

R5 and R7 are independently selected from the group consisting of hydrogen or halo, or

R4 and R5, or R4 and R6 together form a heterocyclic ring condensed with 5 or 6 atoms, optionally substituted with halogen or C ¹ alkyl - ^6; and

A is a fused heterocyclic ring; or a pharmaceutically acceptable salt thereof.

Suitably, C ¹ -C ⁶ alkyl functionalities can be independently selected from the group consisting of: methyl, ethyl, propyl, pentyl and hexyl. The alkyl functionality can be linear or branched. Methyl and ethyl alkyl functionalities are particularly preferred, with methyl being the most preferred alkyl functionality.

The NR1R2 residue of formula IIa can be in the 4 'position or can be in the 3' position. R1 and R2 can be H. R9 can be methyl, ethyl, n-propyl or isopropyl.

Also disclosed herein are compounds that may be represented by

where p is 1 or 2;

R6 is selected from the group consisting of OH or -OR9, where R9 is as defined above; and

R10 is independently selected at each occurrence from the group consisting of H, halogen or C ^1-6, for example, methyl or ethyl.

The disclosure also provides, at least in part, compounds represented by the formulas represented below, for use in the disclosed procedures.

For example, described herein for use in the disclosed procedures are compounds of formula III:

wherein X is C ¹ -C ³ alkylene, optionally substituted with one, two or three substituents selected from halogen or hydroxyl;

Ri is selected from the group consisting of C ¹ -C ^6, cycloalkyl C ³ -C ⁶ alkenyl , C ² -C ⁶ alkynyl , C ² -C ^6; R ² is selected from the group consisting of hydrogen and C ¹ -C ⁶ alkyl;

R ³ is independently selected each occurrence from the group consisting of hydrogen, alkoxy C ¹ -C ⁶ alkyl C ¹ -C ⁶ alkyl, cyano, C ³ -C ⁶ cycloalkyl, halogen, hydroxyl and nitro;

R ⁴ is selected from the group consisting of hydrogen and C ¹ -C ⁶ alkyl;

R ⁵ is C ¹ -C ⁶ alkyl;

or pharmaceutically acceptable salts or N-oxides thereof.

Suitably R ⁵ can be H.

Suitably, C ¹ -C ⁶ alkyl functionalities can be independently selected from the group consisting of: methyl, ethyl, propyl, pentyl and hexyl. The alkyl functionality can be linear or branched. Methyl and ethyl alkyl functionalities are particularly preferred, with methyl being the most preferred alkyl functionality.

R ¹ of formula III can be C ¹ -C ⁶ -alkyl, such as methyl. R ² can be hydrogen. R ³ can be selected from the group consisting of hydrogen, C ¹ -C ⁶ -alkyl, halogen and hydroxyl. R ³ can be hydrogen. R ⁴ and R ⁵ can each be C ¹ -C ⁶ alkyl. R ⁴ can be hydrogen and R ⁵ can be methyl. X can be (CH ² ) n, where n is 1 or 2, such as 1.

The -NR ² -COR ¹ residue of formula III may be in the meta or para position relative to X as shown in formulas IIIa and IIIb.

The disclosure also provides compounds represented by formula IV, as shown below, which can be used in the disclosed procedures, for example:

wherein Ri is selected from the group consisting of C ¹ -C ^6, cycloalkyl C ³ -C ⁶ alkenyl , C ² -C ⁶ alkynyl , C ² -C ^6;

R ² is selected from the group consisting of hydrogen and C ¹ -C ⁶ alkyl;

R ³ is independently selected each occurrence from the group consisting of hydrogen, alkoxy C ¹ -C ⁶ alkyl C ¹ -C ⁶ alkyl, cyano, C ³ -C ⁶ cycloalkyl, halogen, hydroxyl and nitro;

R ⁵ is hydrogen or C ¹ -C ⁶ alkyl;

or pharmaceutically acceptable salts or N-oxides thereof.

The compounds of Formula V are also disclosed as shown below:

wherein R ¹ is selected from the group consisting of C ¹ -C ^6, cycloalkyl C ³ -C ⁶ alkenyl , C ² -C ⁶ alkynyl , C ² -C ^6;

R ³ is independently selected each occurrence from the group consisting of hydrogen, alkoxy C ¹ -C ⁶ alkyl C ¹ -C ⁶ alkyl, cyano, C ³ -C ⁶ cycloalkyl, halogen, hydroxyl and nitro;

R ⁴ is selected from the group consisting of hydrogen and C ¹ -C ⁶ alkyl;

R ⁵ is hydrogen or C ¹ -C ⁶ alkyl; and A is a fused heterocycle of five or six members;

or pharmaceutically acceptable salts or N-oxides thereof.

R ¹ can be C ¹ -C ⁶ alkyl, such as methyl. R ¹ and R ³ can each be C ¹ -C ⁶ alkyl, such as methyl. R ² can be hydrogen.

Also disclosed is a compound that can be represented by

where p is 1 or 2;

R1 is selected from the group consisting of C1-C6 alkyl, C3-C6 cycloalkyl, C2-C6 alkenyl, and C2-C6 alkynyl; R ⁴ and R ^{8 are} each independently selected from the group consisting of hydrogen and C ¹ -C ⁶ alkyl; or pharmaceutically acceptable salts or N-oxides thereof.

For example, the compounds disclosed herein for use in the described procedures include compounds of formula A:

Rio is selected from the group consisting of H and C ¹ -C ⁶ -alkyl (eg methyl);

Ríí is selected from the group consisting of H, C ¹ -C ⁶ -alkyl and -C (O) -C ¹ -C ⁶ -alkyl; (eg R ¹¹ can be H, methyl, -C (O) -methyl or -C (O) -ethyl).

R ⁵ is C ¹ -C ⁶ -alkyl (eg R ⁵ perhaps methyl, ethyl or propyl); or pharmaceutically acceptable salts or N-oxides thereof.

For example, formulas A 'and A "are described herein for use in the disclosed procedures:

Examples of compounds herein for use in the disclosed procedures include:

or a pharmaceutically acceptable salt thereof.

The compounds disclosed herein include: 4-amino-N-hydroxy-2-methoxybenzamide (compound 13); 6-methoxy quinolin-5-carboxylic acid (compound 36); 6-methoxy-1,2,3,4-tetrahydroquinolin-5-carboxylic acid (compound 37); and 5-diisopropylaminosalicylic acid (compound 38).

Other compounds disclosed herein include those represented by:

(compound 13):

(compound 14):

(compound 26):

(compound 17):

(compound 31):

(compound 28):

The compounds disclosed herein include racemic mixtures and enantiomers of compounds, for example: (±) -2-hydroxy-3- (3'-aminophenN) propionic acid (compound 20); (±) -2-methoxy-2- (4'-aminophenyl) acetic acid (compound 23); (±) -2-ethoxy-2- (3'-aminophenN) acetic acid (compound 32); (±) -2-ethoxy-2- (4'-aminophenyl) acetic acid (compound 33); (±) -2-methoxy-3- (4'-aminophenyl) propionic acid (compound 34) "± 34" (racemic form); (±) -2-ethoxy-3- (4'-aminophenyl) propionic acid (compound 39); (±) -2-ethoxy-3- (3'-aminophenyl) propionic acid (compound 40).

For example, the compounds used in the procedures disclosed herein may be enantiomers of the following racemic mixtures: (R, S) -2-hydroxy-2- (3-aminophenyl) acetic acid (compound 10); (R, S) -2-hydroxy-2- (4-aminophenyl) acetic acid (compound 11); (R, S) -2-hydroxy-3- (4'-aminophenyl) propionic acid (compound 21); (R, S) -2-methoxy-2- (3'-aminophenyl) acetic acid (compound 22); (R, S) -2-methoxy-3- (3'-aminophenyl) propionic acid (compound 35); (R, S) -2-methoxy-3- (4-aminophenyl) propionic acid (compound 34), as well as enantiomers, for example: (+) 2-S-methoxy-3- (4-aminophenyl) propionic acid; (-) 2-R-methoxy-3- (4-aminophenyl) propionic acid. In some aspects of the disclosure, (R, S) -2-hydroxy-3- (4'-aminophenyl) propionic acid (compound 21) or (±) -2-hydroxy-3- (3 'acid) is preferred. -aminophenyl) propionic (compound 20).

Other mixtures of racemic compounds of compounds disclosed herein include: eg, (±) -2-hydroxy-2- (3-aminophenyl) acetic acid (compound 10); (±) -2-hydroxy-2- (4'-aminophenyl) acetic acid (compound 11); (±) -2-hydroxy-3- (4'-aminophenyl) propionic acid (compound 21) and (±) -2-methoxy-2- (3'-aminophenyl) acetic acid (compound 22).

Additional compounds disclosed herein for use in the procedures described include: 5-aminosalicylic hydroxamic acid (compound 5); 3-dimethylaminosalicylic acid (compound 6); 2-methoxy-4-aminobenzoic acid (compound 7); 2-methoxy-5-aminobenzoic acid (compound 8); 5-methylaminosalicylic acid (compound 9); 4-methylaminosalicylic acid (compound 12); 4-acetylaminosalicylic acid (compound 16); 2-ethoxy-4-aminobenzoic acid (compound 18); 2-ethoxy-5-aminobenzoic acid (compound 19); 4-dimethylaminosalicylic acid (compound 24); 2-ethoxy-4-aminobenzoylhydroxamic acid (compound 25); 6-hydroxyquinolin-5-carboxylic acid (compound 27); 2- (2-propyl) oxy-4-aminobenzoic acid (compound 30); 4- (1-piperazinyl) salicylic acid (compound 41); (R, S) 5-oxa-quinolin-6-carboxylic acid (compound 15); 6-methoxy quinolin-5-carboxylic acid (compound 36); 6-methoxy-1,2,3,4-tetrahydroquinolin-5-carboxylic acid (compound 37); 5-diisopropylaminosalicylic acid (compound 38); and 4-diisopropylaminosalicylic acid (compound 42).

Compounds and pharmaceutical compositions, comprising at least one compound, may be selected herein from the group consisting of: N-acetyl- (R) - (-) - 3- (4-aminophenyl) -2- acid. methoxypropionic, N-acetyl- (R) - (-) - 3- (4-aminophenyl) -2-methoxypropionic acid; and racemic N-acetyl- (S) - (-) - 3- (4-aminophenyl) -2-methoxypropionic acid; or a compound selected from:

or pharmaceutically acceptable salts or N-oxides thereof.

Other disclosed compounds include compound 20, compound 21,4-acetamino-N-hydroxy-2-methoxybenzamide; 1-acetyl-6-methoxy-1,2,3,4-tetrahydroquinolin-5-carboxylic acid, 5-acetamido-2-hydroxybenzoic acid (eg, acetylated 5-aminosalicylic acid) or pharmaceutically acceptable salts or N-oxides of the same.

Also disclosed herein for use in the disclosed procedures are compositions that include a compound represented by the represented formulas and, for example, a pharmaceutically acceptable carrier.

Procedures for preparing contemplated compounds can be found, for example, in WO2007 / 010516 and WO2007 / 010514, each incorporated herein by reference in its entirety.

Procedures

Procedures for preventing or treating fibrosis, such as liver fibrosis and / or intestinal fibrosis, are part of this disclosure. Such procedures may comprise administering to a patient in need or to a patient at risk, a pharmaceutical preparation comprising a PPARy agent, such as a compound disclosed herein, eg, Formulas I, IIa or IIb, eg, compounds 17, 29, 34 or 39. For example, there is provided a method of preventing or treating liver fibrosis which comprises administering to a patient in need a compound disclosed herein. Alternatively, there is provided a method of preventing or treating intestinal fibrosis which comprises administering to a patient in need a compound disclosed herein.

Patients treated with the above procedure may or may not have detectable fibrosis. The patient may have at least about 5%, 10%, 20%, 30%, 40%, or even 50% or more reduction in the amount of fibrosis present in the patient after administering a disclosed compound, eg, the Compounds 17, 29, 34, or 39, after, for example, 1 day, 2 days, 1 week, 1 month, or 6 months or more. The administration of said compound can be carried out, for example, at least daily. The compound can be administered orally. The delay of the clinical manifestation of fibrosis in a patient as a consequence of the administration of a compound disclosed herein can be at least, for example, 6 months, 1 year, 18 months or even 2 years or more compared to a patient who is not administered a compound such as that disclosed herein.

A needy patient may have liver fibrosis that has turned into cirrhosis. A patient at risk for liver fibrosis may include patients with hepatitis B, hepatitis C, or non-alcoholic steatohepatitis (NASH). NASH is included in the spectrum of non-alcoholic fatty liver diseases, including steatosis and cirrhosis. NASH is a component of metabolic syndrome, which is characterized by obesity, type 2 diabetes mellitus, and dyslipidemia, and can ultimately lead to hepatocellular carcinoma.

Procedures for treating disorders associated with liver fibrosis are also disclosed herein, such as the treatment of at least one of: certain storage diseases and innate errors of metabolism, such as, alpha 1 -antitrypsin deficiency, diseases by copper storage (eg, Wilson's disease), fructosemia, galactosemia, glycogen storage diseases (eg, Types III, IV, VI, IX, and X), iron overload syndromes (eg, hemochromatosis), abnormalities lipids (eg, Gaucher disease), peroxisomal disorders (eg, Zellweger syndrome), and tyrosinemia; bacterial infections (eg brucellosis); parasitic infections (eg, echinococcosis); NASH; viral infections (eg, hepatitis B or hepatitis C, including chronic hepatitis B or C); Budd-Chiari syndrome; heart failure; hepatic veno-occlusive disease; and thrombosis of the portal vein. Procedures for treating congenital liver fibrosis are also disclosed herein. The composition can be administered orally.

Drug and / or alcohol abuse has been implicated in cases of liver fibrosis. Procedures for treating liver fibrosis in a patient with a history of drug and / or alcohol abuse are described herein. For example, A patient with a history of abuse of at least one of the following: alcohol, amiodarone, chlorpromazine, isoniazid, methotrexate, methyldopa, oxyphenisatin, and tolbutamide.

A patient at risk for intestinal fibrosis may include patients with ulcerative colitis, inflammatory bowel disease, or Crohn's disease. A patient at risk may also include patients at an early age at the time of diagnosis of Crohn's disease or colitis, extensive and / or severe colonic disease, patients with the presence of primary sclerosing cholangitis and / or patients with a family history of cancer. .

Procedures for treating disorders associated with intestinal fibrosis are also disclosed, such as treating at least one of: ulcerative colitis, inflammatory bowel disease, or Crohn's disease.

Methods for preventing or treating renal fibrosis, cardiac fibrosis, endomyocardial fibrosis, idiopathic pulmonary fibrosis, myelofibrosis, retroperitoneal fibrosis, or nephrogenic systemic fibrosis are disclosed herein, comprising administering to a patient in need, a pharmaceutical preparation comprising a PPARy agent, such as a compound disclosed herein.

The compounds disclosed herein can be used alone or in combination with each other, where at least two compounds of the disclosure are used together in a single composition or as part of a treatment regimen. The compounds disclosed herein can also be used in combination with other drugs to treat drug and / or alcohol abuse, renal fibrosis, cardiac fibrosis, endomyocardial fibrosis, idiopathic pulmonary fibrosis, myelofibrosis, retroperitoneal fibrosis, or nephrogenic systemic fibrosis, abuse of drugs and / or alcohol.

Generally, a therapeutically effective amount of active component will be in the range of from about 0.1 mg / kg to about 100 mg / kg, optionally from about 1 mg / kg to about 100 mg / kg, optionally from about 1 mg / kg to 10 mg / kg. The amount to be administered will depend on variables such as the type or scope of the disease or indication to be treated, the general state of health of the patient in particular, the relative biological efficacy of the administered binding protein, the formulation of the binding protein , the presence and types of excipients in the formulation and the route of administration. It is understood that the initial dose administered may be increased beyond the upper level in order to rapidly reach the desired blood level or tissue level, or the initial dose may be smaller than the optimal dose and the daily dose may be progressively increased. during the course of treatment according to the specific situation. Human dosage can be optimized, for example, in a conventional phase I dose-increase study designed to go from 0.5 mg / kg to 20 mg / kg. The frequency of dosing can vary, depending on factors such as the route of administration, the amount of dosing, and the disease being treated. Example dosing frequencies are once a day, once a week, and once every two weeks.

The formulations or compositions disclosed herein comprise a disclosed compound and typically include a pharmaceutically acceptable carrier.

The compositions disclosed herein can be administered by various means, depending on their intended use, as is well known in the art. For example, if the compositions of the present disclosure are administered orally, they can be formulated as tablets, capsules, granules, powders, or syrups. Alternatively, the formulations of the present disclosure can be administered parenterally as injections (intravenous, intramuscular, or subcutaneous), drip preparations, or suppositories. For application via the ophthalmic mucous membrane route, the compositions of the present disclosure can be formulated as eye drops or eye ointments. These formulations can be prepared by conventional means, and, if desired, the compositions can be mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrector, a solubilizing agent, an auxiliary to the suspension, an emulsifying agent or a coating agent.

In the formulations of the subject disclosure, wetting, emulsifying and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may be present in formulated agents.

The subject compositions disclosed herein may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol, and / or parenteral administration. The formulations can be conveniently presented in unit dosage form and can be prepared by any of the procedures well known in the art of pharmacy. The amount of composition that can be combined with a carrier material to produce a single dose varies depending on the subject to be treated and the particular mode of administration.

The procedures for preparing the formulations disclosed herein include the step of associating compositions of the present disclosure with the vehicle and, optionally, one or more auxiliary ingredients. In general, the formulations are prepared by uniformly and intimately associating the agents with liquid vehicles or finely divided solid vehicles or both, and then, if necessary, molding the product.

Formulations suitable for oral administration may be in the form of capsules, wafers, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth), powders, granules or as a solution or suspension in an aqueous or non-aqueous liquid, or as a liquid oil-in-water or water-in-oil emulsion, or as an elixir or syrup, or as tablets (using an inert base, such as gelatin and glycerin, or sucrose and gum arabic) , each containing a predetermined amount of a composition subject thereto as the active ingredient. The compositions of the present disclosure can also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, film-coated tablets, sugar-coated tablets, powders, granules, and the like), the subject composition is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or expanders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or gum arabic; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type can also be used as fillers in hard and soft filled gelatin capsules, using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols, and the like.

The formulations and compositions can include micronized crystals of the disclosed compounds. Micronization can be performed on crystals of the compounds alone, or on a mixture of crystals and some or all of pharmaceutical excipients or carriers. The average particle size of the micronized crystals of a disclosed compound can be, for example, from about 5 to about 200 microns, or from about 10 to about 110 microns.

A tablet can be made by compression or molding, optionally with one or more auxiliary ingredients. Compression tablets can be prepared using a binder, (eg, gelatin or hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (eg, sodium starch glycollate or cross-linked sodium carboxymethyl cellulose), surfactant, or dispersant. Molded tablets can be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets and other solid dosage forms, such as film-coated tablets or sugar-coated tablets, capsules, pills and granules, can optionally be scored or prepared with coatings and coatings, such as enteric coatings and other coatings well known in the art of the pharmaceutical formulation.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the subject composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, acetate ethyl, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular cotton, peanut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid sorbitan esters, cyclodextrins, and mixtures thereof.

Suspensions, in addition to the subject composition, may contain suspending agents such as, for example, isostearyl ethoxylated alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth gum, and mixtures of the same.

Formulations for rectal or vaginal administration can be presented as a suppository, which can be prepared by mixing an object composition with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate. , and which is solid at room temperature, but liquid at body temperature and will therefore melt in the body cavity and release the active agent. Formulations that are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such vehicles as are known in the art to be appropriate.

Dosage forms for transdermal or topical administration of an object composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable vehicle and with preservatives, buffers, or propellants, as may be necessary.

Ointments, pastes, creams and gels may contain, in addition to an object composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays may contain, in addition to an object composition, excipients, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Aerosols may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

The compositions and compounds of the present disclosure can alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, a liposomal preparation, or solid particles containing the compound. A nonaqueous suspension (eg, fluorocarbon propellant) could be used. Nebulizers can be used sonic because they minimize exposure of the agent to shear, which can result in degradation of the compounds contained in the subject compositions.

Generally, an aqueous aerosol is manufactured by formulating an aqueous suspension or solution of an object composition together with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary with the requirements of the particular subject composition, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), harmless proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers , salts, sugars or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

The pharmaceutical compositions of the present disclosure suitable for parenteral administration comprise a composition disclosed in combination with one or more sterile pharmaceutically acceptable aqueous or non-aqueous isotonic solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into just sterile injectable solutions or dispersions. prior to use, they may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the intended recipient's blood, or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous vehicles that can be used in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil and injectable organic esters, such as ethyl oleate and cyclodextrins. Adequate fluidity can be maintained, for example, by using coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. The effectiveness of treatment with the subject compositions can be determined in various ways known to those skilled in the art.

Throughout the description, when it is described that the compositions have, include or comprise specific components, it is contemplated that the compositions also consist essentially of, or consist of, the cited components. Similarly, when the processes are described as having, including or comprising specific process steps, the processes also essentially consist of, or consist of, the aforementioned processing steps. Except where otherwise indicated, the order of the stages or the order to perform certain actions are immaterial as long as the disclosure remains operable. Furthermore, unless otherwise indicated, two or more stages or actions may be carried out simultaneously.

Examples

The disclosure is illustrated in more detail in the following examples. The following examples are provided for illustrative purposes only, and should not be construed as limiting the scope or content of the disclosure in any way.

Example 1: Study of liver fibrosis

Liver fibrosis was induced in male C57bl6 mice (Charles River, l'Arbresle, France) by injection of carbon tetrachloride (CCU). C57bl6 mice weighed 22-25 g and were kept under laboratory conditions for 1 week before the experiment. Animals were housed 5 per cage with food and water available ad libitum.

The study consisted of 5 test groups (see Table 1): control probe (CG), intraperitoneal control (CIP), carbon tetrachloride (CCU), compound 34 and agent JWH-133 (see Julien et al., ( 2005) Gastroenterology 128: 742-755 (for JWH-133, a specific CB2 agonist and an antifibrogenic agent)).

TABLE 1

continuation

The effectiveness of compound 34 (PPARg-specific ligand) was evaluated in comparison with JWH-133 (antifibrogenic agent). Compound 34 was administered orally in combination with CCU injections for approximately 5 weeks. Compound 34 was dosed daily at 200 µl of a 30mM solution. JWH-133 was administered daily through intraperitoneal injections at 3 mg / kg (dose volume = 100 pl). The liver fibrosis pattern is induced by chronic exposure to increased doses of CCU for 5 weeks. The first week, the mice received an intraperitoneal injection of CCU, resuspended in olive oil, at the dose of 80 jl / kg body weight of mice twice a week. At week 2, 3 CCU injections were made at the 160 jl / kg dose every other day. At week 3, 3 CCU injections were made at the 240 jl / kg dose every other day. At week 4, 3 injections of CCU were made at the dose of 320 jl / kg every other day. Mice were sacrificed at week 5, 3 days after the last CCU administration.

Body weights were recorded for the duration of the experiment. As seen in figure 1, no differences in the evolution of body weight were detected between the different groups of mice.

The livers were excised and the aliquots were frozen in liquid nitrogen and kept at -80 ° C until analysis. A portion of each part of the liver was fixed in 10% formalin for histology.

Example 2: Histological analysis of the liver fibrosis study

Formalin-fixed liver tissue was processed and 5 jm thick paraffin sections were stained with hematoxylin and eosin (H&E) and Picrosirius red staining (collagen specific staining in tissue sections) to assess the degree of fibrosis in the liver . Each section was blindly analyzed by 3 different experimenters for quantification of the degree of fibrosis for each lobe of the liver (5) using the Metavir score (see Figure 2A for the Metavir score table). The photographs shown in figure 2B were obtained with a 20x objective. The control group had an F0 score and the CCU group had an F3 score, indicating fibrosis of the liver. Compound 34 (GED) showed a greater reduction in fibrosis than the JWH-133 group with respective scores of F1 for Compound 34 and F2 for JWH-133.

As expected, fibrosis was not observed in the two groups of control mice (control probe (CG) and control IP (CIP)). In the group of fibrotic mice (CCU group) receiving vehicle only, an average score of 2.5 ± 0.5 was observed corresponding to moderate to severe fibrosis according to the Metavir score (see Figure 3A). For fibrotic mice receiving JWH-133 or compound 34 (GED), a score of 2 ± 0 and 2.1 ± 0.2, respectively, was observed. A decrease in fibrosis grade from severe to moderate fibrosis was observed with similar efficacy for both JWH-133 and GED.

These results were confirmed using an adapted Metavir score (FIG. 3B): each section of the liver was blindly analyzed by 2 investigators for low magnification (X5) of> 30 portal pathways classified from F0 to F4 according to the MeTAVIR score (Score (of 1 section) = $ (PTF1 PTf2 PTF3 PTF4)). Using this score, it was shown that JWH and GED decrease respectively 14.2% and 12.5% of the fibrotic score compared to untreated animals and that the therapeutic effect of the 2 tested molecules seems similar.

Example 3: Analysis of gene expression from the liver fibrosis study

The effect of administration of compound 34 (GED) and CB2-specific agonist (JWH-133) was evaluated on the level of gene expression of cytokines (IL-1 p, TNF-a) and genes involved in the mechanism of fibrosis by quantitative real-time PCR.

Total RNA was isolated from the livers using the Rneasy kit (Macherey Nagel, Hoerdt, France) according to the manufacturer's instructions. RNA quantification was performed by spectrophotometry. After treatment at 37 ° C for 30 minutes with 20-50 units of RNase-free DNase I (Roche Diagnostics Corporation, Indianapolis, IN, USA), THE oligo-dT primers (Roche Diagnostics Corporation, Indianapolis, USA. ) Were used to synthesize single-stranded cDNA. MRNAs were quantified using SYBR Green Master Mix (Applera, Courtaboeuf, France) with murine specific oligonucleotides (see Table 2) on a GeneAmp Abiprism 7000 (Applera, Courtaboeuf, France). Calibrated and template-free controls were included in each assay. Each sample was processed in triplicate. The SYBR green tint intensity was analyzed using Abiprism 7000 SDS software (Applera, Courtaboeuf, France). All results were normalized with respect to the unaffected domestic p-actin gene.

TABLE 2

There was no significant modification of cytokine gene expression in the different groups of mice. Regarding the genes involved in the fibrosis mechanism, an increase in the expression of □ -SMA and collagen-1 was observed in animals with fibrosis (CCU group) compared to control animals (CG and CIP groups). As seen in Figure 4 and Table 3, there was a decrease in the level of α-SMA and collagen-1 in the GED and JWH-133 mice groups compared to the CCl ⁴ group (fibrosis control). For the collagen-1 level, the decrease was significant for the group of mice treated with compound 34 compared to CCU mice with, respectively, 42.81 ± 14.81, p = 0.03 vs. 75.15 ± 5.23. These biological data along with the histological improvement observed in mice treated with compound 34 suggest that compound 34 may have therapeutic antifibrotic properties.

TABLE 3

Example 4: Analysis of liver blood enzymes from liver fibrosis study

The effect of oral administration of compound 34 (GED) and specific CB2 agonist (JWH-133) was evaluated on different hepatic biochemical parameters in the serum of mice: liver enzymes (alanine aminotransferase (AST) and aspartate aminotransferase (ALT)) , Gamma GT and alkaline phosphatase.

As expected, 4 days after CCU administration, with no improvement in AST, ALT and Alk P were observed in any group of mice. Compound 34 was not associated with any improvement in liver blood enzymes suggesting an absence of liver toxicity.

As evaluated by histology and liver markers, JWH-133 and Compound 34 have similar antifibrotic effects in mice with liver fibrosis induced by repeated injections of CCU. As expected, JWH decreases liver fibrotic lesions by 14% and normalizes the liver concentration of collagen-1 mRNA. Compound 34 has a similar effect without liver toxicity. The data suggests that compound 34 may be the first compound with both anti-inflammatory and antifibrotic properties.

Example 5: Study of intestinal fibrosis

This example describes an experiment to evaluate the effect of compound 34 on intestinal fibrosis. Chronic colitis was induced in C57bl6 mice in 3 cycles of 2.5% DSS (40,000-50,000 MW, TdB Consultancy AB, Sweden) dissolved in sterile drinking water for 5 days, followed by 7 days of regular drinking water.

The study consisted of 3 test groups (see Table 4): control group, DSS control group, GED group (compound 34).

TABLE 4

continuation

Compound 34 was used in its optimal dose (30 mM) and administered by oral gavage daily after the second cycle of DSS and until sacrifice. Mice receiving DSS were checked twice weekly for the development of colitis by monitoring body weight. Euthanasia of the mice was performed one week after the third cycle of DSS (see Figure 5). The evaluation of the size and weight of the colon was evaluated postmortem.

Example 6: Analysis of the intestinal fibrosis study

A. Colon weight / size ratio

Intestinal fibrosis is characterized by a shortening and thickening of the colon and adhesions. The measurement of the weight / size ratio of the colon is an indicator of the level of inflammation and fibrosis. As seen in Figure 6A, a significant 124% increase in colon weight / size ratio was observed in DSS mice receiving the vehicle compared to control mice receiving only regular water, with, respectively, 44 , 98 ± 6.31 vs. 20.11 ± 3.91, p <0.05. These results are illustrated by a significant shortening of the colon in DSS mice (Figure 6B). Compound 34 causes a 34% reduction in the profibrotic effect of DSS, leading to a significant decrease in colon weight / size ratio of 38.13 ± 7.82 vs. 44.98 ± 6.31 (p <0.05) compared to vehicle receiving DSS mice (FIG. 6A). Consequently, it reduces the morphological signs of fibrosis, such as shortening and thickening of the colon, and decreases collagen deposits in the colon (FIG. 6B).

B. Inflammation level estimated by histology

Samples were taken from the rings of the transverse part of the colon (postmortem) and fixed in 4% formaldehyde and embedded in paraffin for histological analysis. Sections (4 pm) were stained with May-Grunwald-Giemsa staining and two investigators performed a multiparameter histological score (0 to 18) in a masked manner. This staining allowed the quantification of inflammation. Histological grades, which reflect the level of inflammation, were assigned depending on the intensity of the cellular infiltrate in the mucosa, its extension in the submucosal layers and the presence of epithelial lesions (see Table 5).

TABLE 5

Segment Grade Description

Severity 0 None

1 Light

22 Moderate

3 Serious

scope 0 none

1 mucosa

2 Mucosa and submucosa

3 Transmural

Regeneration 4 No tissue repair

3 Non-intact superficial epithelium

₉ Regeneration with depletion of

crypts

¹ almost complete regeneration

⁰ Complete regeneration or normal tissue

Crypt damage ⁰ None

¹ Basal 1/3 damaged

² Basal 2/3 damaged

³ Only intact superficial epithelium

⁴ Loss of all crypt and epithelium

Impact percentage ¹ 1-25%

² 26-50%

(continuation)

Segment Grade Description

4 76-100%

Inflammatory status was evaluated histologically for each colon of mice using a classic MGG stain. As expected, significant inflammation of 8.4 pt and strong was observed in the group of mice that received the DSS compared to the control mice that received normal water, with, respectively, 3.77 ± 0.80 against 0.50 ± 0.33, p <0.05 (see FIG. 7). This result confirms the induction of inflammation with the 3 cycles of DSS. A significant decrease in the level of inflammation was observed in DSS mice receiving compound 34 compared to DSS mice receiving vehicle alone with, respectively, 2.18 ± 0.48 versus 3.77 ± 0.80, p <0.05.

C. Fibrosis level

C1. Picrosirius red staining evaluation

The obtained colon samples were rapidly fixed with 10% buffered formalin in PBS (pH 7.4) for 3 hours, dehydrated in graded ethanol and embedded in low temperature melting paraffin. Formalin fixed colonic tissue was processed and 5 µm thick paraffin sections stained with Picrosirius red stain for histological analysis. Images were obtained with an x20 objective. Each section was analyzed in a masked manner by 3 different experimenters to quantify the degree of fibrosis using a score from 0 to 4.

A significant increase in colon fibrosis level was observed in DSS mice compared to control mice with 2.27 ± 0.12 vs. 1.12 ± 0.07 respectively (see Figure 8). This result confirms that the chronic inflammation induced by 3 cycles of d Ss induces intestinal fibrosis. It also confirms that the administration of compound 34 to DSS mice decreases the level of fibrosis compared to DSS mice receiving the vehicle alone (1,625 ± 0.15 vs. 2.27 ± 0.12, p <0.05).

C2. Evaluation using Masson's trichrome stain

In this evaluation of the level of colonic fibrosis, a Masson's trichrome stain and collagen were used. The obtained colon samples were rapidly fixed with 10% buffered formalin in PBS (pH 7.4) for 3 hours, dehydrated in graded ethanol and embedded in low temperature melting paraffin. Serial sections of 3 pm were incubated for 40 minutes in methanol and 3% hydrogen peroxide solution, and then rinsed in PBS. Intestinal fibrosis was classified as absent, mild or severe, depending on the density and extent of staining of the trichrome-positive connective tissue and the alteration of tissue architecture.

C3. Assessment of collagen levels

For immunohistochemical analysis, colon tissue samples were fixed in fresh 4% paraformaldehyde (PFA) / PBS solution for 3 hours at room temperature, dehydrated in serially graded ethanol, and embedded in low temperature fusion paraffin. . 3 pm thick sections were incubated in methanol for 40 minutes and then in 3% hydrogen peroxide for 5 minutes. The samples were incubated overnight with specific antibodies against collagen types I-III (Abcam), connective tissue growth factor (CTGF) (Abcam), platelet derived growth factor (PDGF), SMAD2 / 3, the main markers of fibrosis. The samples were washed for 5 minutes with PBS and incubated with streptavidin-biotin-peroxidase conjugated secondary antibody (Dako LSAB Corporation, cod K0675, Dako-Cytomation, Milano). After washing in PBS for 10 minutes, the sections were incubated with 3,3-diaminobenzidine-tetrahydrochloride for 1-3 minutes.

The specificity of the immune reaction was performed by omitting the primary antibodies. Finally, the samples were stained with Mayer's hematoxylin and viewed with the Olympus BX51 Light Microscope (Olympus, Optical Co. Ltd., Tokyo, Japan).

In immunohistochemical analysis (x 20) of collagens I-VII in colon of DSS and control mice, staining of collagens I-VII in the large intestine of DSS mice was mainly located in the connective tissue of the submucosa and the muscularis propria, where strong staining of collagen was observed (see Figure 9). As shown in Figure 9, the administration of compound 34 is associated with a decrease in collagen staining indicating that it has improved fibrosis and reduces the development of intestinal fibrosis.

In addition to anti-inflammatory effects, administration of compound 34 rapidly decreases the molecular events leading to fibrosis and prevents DSS-induced intestinal fibrotic lesions in mice.

For Western blot analysis, 0.5 cm of frozen colon samples were homogenized in RIPA buffer containing 50 mM Tris HCl pH 7.6, 150 mM NaCl, 1.5 mM MgCh, 5 mM EDTA, 1% Triton -X and 10% glycerol, supplemented with 100 mM sodium fluoride (NaF), 2 mM sodium orthovanadate (Na ³ VO ⁴ ), 10 mM sodium pyrophosphate (NaPPi), 1 mM phenylmethanesulfone (PMSF) and a classic cocktail of protease inhibitor containing 10 | jg / ml leupeptin and aprotinin.

For each sample, 30 jg of protein was separated by PAGE and electrotransferred. Nitrocellulose membranes (100% pure) were incubated (according to a specific protocol for each antibody) with primary antibodies directed against CTGF, Collagen-I and GAPDH (purchased from Abcam, Cambridge, UK; 1: 1000 for 2 hours at RT), diluted in 5% skim milk in 0.1% TBS-t. The membranes were subsequently washed in 0.1% TBS-t and incubated with horseradish peroxidase conjugated secondary antibodies (anti-rabbit and anti-mouse; Sigma Aldrich; 1: 20,000 for 1 hour at room temperature) diluted in 5% skim milk in 0.1% TBS-t. Finally, immunodetection was performed with the chemiluminescent substrate SuperSignal West Pico (Thermo Scientific Pierce, Erembodegem) according to the manufacturer's protocol. The membranes were exposed to the autoradiography film (Fuji Photo Film Co., Dusseldorf, Germany). The optical density of the target bands was determined using a computer-aided densitometer and ImageJ public domain software (WS, Rasband, ImageJ, US National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov / ij /, 1997-2011). Tissue levels were expressed as optical density units (OD) per total protein amount, normalized with internal GAPDH control and the results were expressed as a percentage of the control groups.

Using the IHC procedures and specific antibodies, a reduction of the main fibrosis markers (collagen I-III, CTGF, SMAD 2/3, PDg F, a-SMA and TGF-p1) was observed in the colon of DSS mice that had received the GED compared to colitic mice that received the vehicle (see Figure 9A-9E).

These results were confirmed using a Western blot procedure and different fibrosis markers were quantified in whole colon samples (see Figure 10). As seen in Table 6, a significant decrease in collagen I, CTGF, and IL-13 expression was observed in colitic mice treated with GED compared to colitic mice receiving the vehicle alone (Collagen I 90.75 ± 19.91 vs. 284.4 ± 63.86, p = 0.012, for CTG 55.75 ± 17.53 vs. 115.1 ± 16.88, p = 0.029 and IL-13 88.88 ± 14, 01 vs. 189.5 ± 28.71, p = 0.007).

TABLE 6

Example 7: Study of intestinal fibrosis

This example describes an experiment to evaluate the effect of compound 34 (GED) in the treatment of intestinal fibrosis.

Materials and procedures

In vitro experiments

A total of 60 wild-type C57BL / 6 mice, purchased from Janvier (Le Genest-St-Isle, France), were included in the study. All mice were kept in a specific pathogen-free facility of the Pasteur Institute in Lille (France). Animal experiments were performed in accordance with government guidelines No. 68/609 / EEC.

Induction of chronic colitis

Chronic colitis and fibrosis were induced in mice by oral administration of 2.5% (w / v) DSS in drinking water, in three cycles (5 days of DSS, 7 days of water). Animals were monitored daily for food and fluid intake and weighed at the start of the study and subsequently regularly every three days.

Experimental design

Mice were randomly divided into two groups: i. DSS and ii. DSS GED. Each group consisted of 25 mice and was compared to 10 control animals, which received only water. GED (30 mg / kg / mouse) was dissolved in a solution containing 0.5% carboxymethyl cellulose sodium salt (CMC; MW: 90,000 Da; Sigma Aldrich) and 1% Tween 80 and administered daily by gavage oral (100 jl / mouse). The GED was administered at the beginning of the second cycle.

Colitis Assessment Course

Animals were observed daily for fluid intake, weight changes, and examined for signs of colitis, including weight loss, diarrhea, bleeding, and rectal prolapse, as well as signs of systemic inflammation, such as piloerection, lethargy, and periorbital exudates.

Sample recovery and preparation

Finally, seven days after the last administration of the DSS cycle, the animals of each group were sacrificed by cervical dislocation with deep CO ² anesthesia and underwent a laparotomy. The colon was visualized and quickly removed. The length of the entire colon-rectum and the weight of the 8 cm distal colon-rectum were measured.

Evaluation of macroscopic and microscopic colonic lesions

Macroscopic colonic lesions, including dilation, thickening, and stenosis, were scored by an independent observer, who was unaware of the treatment of the mice. Tissue samples were removed from the colon and immediately frozen or fixed in 4% formaldehyde / PBS solution for 3 hours at room temperature, followed by the standard procedure for paraffin embedding. Paraffin embedded tissues cut crosswise at 3 pm were stained with hematoxylin / eosin, to assess the degree of inflammation, and Masson's trichromatic stain to better assess connective tissue and fibrosis. The stained sections were then masked evaluated with an Olympus BX51 Light Microscope (Olympus, Optical Co. Ltd., Tokyo, Japan) by two pathologists who have performed histological scoring, evaluating: i. presence of ulcers (0 = absent, 1 = small ulcers, 2 = large ulcers); ii. the inflammatory degree (0 = absent 1 = mild, 2 = moderate and 3 = severe); iii. lesion depth (0 = absent, 1 = lesions extending into the submucosa, 2 = lesions in the muscolaris propria and 3 = lesions in the serosa); iv. fibrosis grade (0 = absent 1 = mild, 2 = moderate and 3 = severe). The degree of intestinal inflammation was assessed according to the density and extent of the chronic inflammatory infiltrate, loss of goblet cells, and thickening of the intestinal wall. A total microscopic score was obtained as the sum of all scores (maximum possible score = 10).

Intestinal fibrosis was classified as absent, mild or severe, depending on the density and extent of trichrome-positive connective tissue staining and alteration of tissue architecture.

Immunohistochemistry ica

The colon tissue samples, dissected and fixed in fresh 4% formaldehyde (FA) / PBS solution for 3 hours at room temperature, were dehydrated in serially graded ethanol and embedded in low-temperature fusion paraffin. Sections 3 pm thick were incubated in a solution of methanol and 3% hydrogen peroxide for 45 minutes.

Samples were incubated overnight with antibodies specific for smooth muscle actin a (a-SMA, Abcam), type I-III collagen (Abcam), TGF-p1 (Abcam), connective tissue growth factor (CTGF ) (Abcam), pSmad3 and Smad3 (Cell Signaling), Smad7 (Imgenex) PPAPy (Cell Signaling). The samples were washed for 5 minutes with PBS and incubated with streptavidin-biotin-peroxidase conjugated secondary antibody (Dako LSAB Corporation, cod K0675, Dako-Cytomation, Milano). After washing in PBS for 10 minutes, the sections were incubated with 3,3-diaminobenzidine-tetrahydrochloride for 1-3 minutes. Nucleus counter staining was obtained by hematoxylin eosin. The specificity of the immune reaction was performed while maintaining the primary antibodies. Finally, the samples were stained with Mayer's hematoxylin and viewed under the Olympus BX51 Light Microscope (Olympus, Optical Co. Ltd., Tokyo, Japan).

Western blot analysis

0.5 cm of frozen colon samples were cut and mechanically homogenized in RIPA buffer containing 50mM TrisHCl, pH 7.6, 150mM NaCl, 1.5mM MgCh, 5mM EDTA, 1% Triton-X and 10% glycerol, supplemented with 100 mM sodium fluoride (NaF), 2 mM sodium orthovanadate (Na ³ VO ⁴ ), 10 mM sodium pyrophosphate (NaPPi), 1 mM phenylmethanesulfone (PMSF) and a classic inhibitor cocktail of the protease containing 10 pg / ml of leupeptin and aprotinin.

30 pg of protein was separated for each sample by PAGE and electrotransfer was performed. 100% pure nitrocellulose membranes were incubated (according to a protocol specific to each antibody) with primary antibodies directed against TGF-p1, CTGF, a-SMA, Collagen-I and GAPDh (purchased from Abcam, Cambridge, UK; 1 : 1000 for 2 hours at RT) and IL-13 (purchased online from Antibodies online; 1: 1000 for 2 hours at RT), diluted in 5% skim milk in 0.1% TBS-t. The membranes were subsequently washed in 0.1% TBS-t and incubated with horseradish peroxidase conjugated secondary antibodies (anti-rabbit and anti-mouse; Sigma Aldrich; 1: 20,000 for 1 hour at room temperature) diluted in skim milk 5% in TBS-t 0.1%. Finally, immunodetection was performed with the SuperSignal West Pico chemiluminescent substrate (Thermo Scientific Pierce, Erembodegem) according to the manufacturer's protocol. The membranes were exposed to the autoradiography film (Fuji Photo Film Co., Dusseldorf, Germany). The optical density of the target bands was determined using a computer-aided densitometer and ImageJ public domain software (WS, Rasband, ImageJ, US National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov / ij /, 1997-2011). Tissue levels were expressed as units of optical density (OD) per total protein amount, normalizing with internal GAPDH control and the results were expressed as a percentage of control groups.

In vivo experiments

Cell cultures

The human colon cancer cell line HT-29 (ATCC HTB-38) and intestinal colon fibroblasts (human ATCC CDD-18 co) were grown in Dulbecco's Modified Eagle's Medium (DMEM) and alpha-modified Eagle's Medium, respectively, 100 U / ml of penicillin and 100 pg / ml of streptomycin supplemented with 10% fetal bovine serum (FBS). Cell cultures were maintained in a humidified atmosphere of 95% air and 5% CO ² at 37 ° C.

Generation of cells with silenced PPARy

HT29 were obtained with PPAPy silenced using the pSUPER.retro system (OligoEngine). The forward and reverse target sequences corresponding to nucleotides 105-123 of human PPAPy mRNA (5'-GCCCTTCACTACTGTTGAC-3 '(SEQ ID NO: 11)) were cloned into the BglII / XhoI restriction sites of the pSUPERretro (pRS) vector , giving the product ShPPAR. A negative control pRS plasmid containing the 5-ACGCTGAGTACTTCGAAAT-3 'sequence (SEQ ID NO: 12) directed against the luciferase gene (construct hLuc) was also generated. Both constructs were transfected into HT-29 and Caco-2 cells using AmaxaBiosystems Nucleofector technology, according to the manufacturer's protocol. Stably transfected clones were selected 24 hours after transfection with complete culture medium supplemented with puromycin (5 pg / ml). Silencing of PPARg expression was verified by quantitative RT-PCr and Western blot analysis. Once established, the ShPPAR and ShLuc cell lines were maintained in complete medium supplemented with 25 pg / ml puromycin.

Experimental design

The fibrotic phenotype was induced by stimulating hCCD-18 and HT29 with TGF-p Ing / ml and 10 ng / ml for 4 days, respectively, dissolved in serum-free medium. 1 mM for GED (195.22 g / mol) was administered during the differentiation period. Furthermore, the possible dependence of PPAPy activation has been investigated using GW9662, a specific antagonist of this receptor. GW9662 10.5 M has been administered 4 hours previously to begin TGFp-induced differentiation.

Quantitative RT-PCR

Total RNA was extracted with a Nucleospin RNA kit (Macherey-Nagel, Hoerdt, France). After RNase inactivation, total RNA was cleaned of trace genomic DNA through DNase treatment and eluted without RNase, water without DEPC. RNA purity was assessed by UV spectroscopy on a 220 to 350 nm Nanodrop system and profiled on an Agilent 2100 bioanalyzer. One pg of total RNA was used to perform quantitative RT-PCR using LightCycler-FastStart DNA Master SYBR Green I Roche Diagnostics (Indianapolis, IN) according to manufacturer's protocol.

results

In vivo experiments

Clinical and macroscopic findings on chronic colitis in mice

Chronic oral administration of DSS induced loss of body weight in all chronic mice from the last day of the first DSS cycle (day 5). On day 10, mice receiving DSS showed a lower body weight compared to control mice with, respectively, 23.88 ± 0.31 vs. 25.27 ± 0.35 p <0.05 (Fig. 11 )

Moderate to severe diarrhea was observed in 70% of the DSS-treated mice and 50% of the GED mice on the seventh day of oral DSS administration. In the same period, systemic manifestations, such as piloerection, periorbital exudates and / or lethargy, were observed in 40% of DSS mice and 30% of GED mice. Obvious rectal bleeding or rectal prolapse was not observed at any stage. Mice were sacrificed on day 15 and day 30, respectively, and the colon underwent macroscopic evaluation by measuring the weight of the colon (based on the last 8 cm of the distal large intestine) and the length of the colon and observing the presence adhesions, contractions, dilation, thickness that was rated 0 if absent; 1 if it is mild or moderate; and 2 if it is serious. The total macroscopic score was calculated as the sum of all these scores.

Table 7 summarizes the effects of ongoing GED administration and macroscopic findings of chronic DSS-induced colitis in mice, indicating that GED improves macroscopic lesions in chronic DSS-induced colitis and reduces morphological signs of fibrosis.

All the macroscopic characteristics observed in the DSS treatments showed, in both models, a significant variation that indicated an effective capacity of DSS to induce severe tissue damage. GED treatment improved all macroscopic lesions, which determined significant differences between DSS and DSS plus total macroscopic scores of g Ed. Colon weight and colon weight / body weight ratio showed no significant variation after GED treatment, while colon length seemed restored significantly. Direct observation of the colon did in fact show evident shortening or dilation in most samples treated with DSS and a remission of these characteristics in mice treated with GED.

TABLE 7. Effect of ongoing GED administration and macroscopic findings of chronic colitis

Thus, the weight / size ratio of the colon was used as an additional indicator of the level of inflammation and fibrosis. A significant 124% increase in proportion was observed in mice receiving DSS compared to control mice receiving regular water only, with respectively 44.98 ± 6.31 vs. 20.11 ± 3.91 (p <0.05). GED led to a significant decrease in weight / colon size ratio compared to DSS mice (38.13 ± 7.82 vs. 44.98 ± 6.31 (p <0.05)), causing a reduction 26% with respect to the fibrotic effect of DSS. Consequently, GED can reduce the morphological signs of fibrosis, such as shortening and thickening of the colon.

M icroscopic findings in chronic colitis LA

Various histological parameters were scored according to the intensity of the cellular infiltrate in the mucosa, the extent of inflammation in the submucosal layers and the presence of epithelial lesions and collagen deposition. Significant and strong inflammation was observed in the group of mice that received the DSS compared to the control mice that received regular water. The histological characteristics in both models showed ulceration and degeneration of the mucosa, decrease in goblet cells, extensive inflammatory cell infiltration, and submucosal edema. All of these findings seemed more emphasized in chronic DSS-induced colitis and extensive thickening of the colon wall was evident. Furthermore, the chronic model showed an obvious regeneration of the crypts and a possible spontaneous recovery of the inflammatory degree in long-term treatments (Fig. 12).

The fibrosis level was observed using Masson's trichrome stain, specific for collagen deposition. A significant increase in fibrosis level was observed not only in the colon of DSS mice compared to control mice, but also between the two treatments (Fig. 12). This evidence, together with the observation of a lower inflammatory degree, seems consistent with the postulate that fibrosis starts from an inflammatory response but can subsequently progress independently.

Total microscopic scores were assigned and it was demonstrated that the high inflammatory degree and fibrosis levels, induced by 1 and 3 cycles of DSS, decreased significantly after daily oral administration of GED (3.36 ± 0.55 for GED vs. 6.45 ± 0.82 for DSS, p <0.01) (Figure 12).

Assessment of tissue levels of the main fibrosis markers

Tissue levels of the main fibrosis markers in the chronic DSS-induced colitis model were evaluated by immunohistochemical analysis and confirmed by immunoblotting. Thus, a marked reduction of collagen I-III (Fig. 13a) and a-SMA (Fig. 13b), the specific markers of fibroblast differentiation, were observed in colon of mice undergoing oral administration of treatment with GED, compared to mice that only received DSS. Expression levels of collagen I-III and a-SMA were measured in 6 control mice, 8 DSS mice and 8 DSS GED mice. Both immunochemistry and Western blotting showed that all major fibrosis markers were significantly increased in the group of mice that received DSS compared to control mice that received regular water, and that daily administration of GED is associated with their expression. reduced. The expression levels of each protein were expressed as a percentage of the mean of the control group and each marker of interest was normalized with a cleaning protein such as GAPDH. A marked increase in collagen I-III expression (184%) was observed in DSS mice compared to the control group, as well as a moderate upregulation of 25% for a-SMA. The Oral administration of GED in mice with chronic DSS-induced colitis caused a reduction in the expression levels of these proteins, more significantly marked for collagen I-III (90.75% ± 19.9% for GED vs. 284.4 % ± 63.8% for DSS, p <0.005).

GED controls the TGFp / Smad pathway and its specific activator, IL13.

The ability of GED to control the TGFp / Smad pathway at different levels was observed by immunohistochemical analysis and demonstrated a marked reduction of the different members involved in this pathway, such as TGF-p1, Smad3, CTGF (data not shown).

The benefit of GED in preventing or treating intestinal fibrosis in relation to variations in IL-13 expression levels was observed in Fig. 14a). IL-13 is a specific activator of the TGF p / Smad pathway and this aspect was confirmed by the consequent decrease in the expression levels of TGF-p1 (Fig. 14b) and CTGF (Fig. 14c). Increased IL-13 expression in DSS mice was significantly restored by oral administration of GED with 88.88% ± 14.01% for GED versus 189.5% ± 28.71% for DSS, p <0 , 01. The consequent reduction in TGF-p1 and CTGF expression levels were confirmed by immunoblot analysis, with marked significance for CTGF (55.57% ± 17.53% for GED versus 115.1% ± 16.88% for DSS, p <0.05). Expression levels were expressed as a percentage of optical density compared to the control group and normalized with GAPDH.

Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not as limiting of the invention described herein. Therefore, the scope of the invention is indicated in the appended claims and not in the previous description and, therefore, it is intended that all changes that come within the meaning and scope of the claims are included in them.

Claims (14)

1. The 2-methoxy-3- (4'-aminophenyl) propionic acid compound (compound 34) represented by the formula:

or a pharmaceutically acceptable salt or stereoisomer thereof, for use in the treatment of fibrosis, wherein fibrosis is selected from intestinal fibrosis or idiopathic pulmonary fibrosis. 2. The compound for use according to claim 1, wherein the compound is (S) -2-methoxy-3- (4'-aminophenyl) propionic acid, or a pharmaceutically acceptable salt thereof. 3. The compound for use according to claim 1, wherein the compound is (R) -2-methoxy-3- (4'-aminophenyl) propionic acid, or a pharmaceutically acceptable salt thereof. 4. The compound for use according to any one of claims 1-3, wherein the fibrosis is intestinal fibrosis. 5. The compound for use according to any one of claims 1-3, wherein the fibrosis is idiopathic pulmonary fibrosis. 6. A pharmaceutical preparation comprising a therapeutically effective amount of 2-methoxy-3- (4'-aminophenyl) propionic acid (compound 34), or a pharmaceutically acceptable salt or stereoisomer thereof, and one or more pharmaceutically acceptable carriers, for use in the treatment of a patient suffering from fibrosis, in which fibrosis is selected from intestinal fibrosis or idiopathic pulmonary fibrosis. 7. The pharmaceutical composition for use according to claim 6, the pharmaceutical preparation comprising a therapeutically effective amount of (S) -2-methoxy-3- (4'-aminophenyl) propionic acid, or a pharmaceutically acceptable salt thereof. . The pharmaceutical composition for use according to claim 6, the pharmaceutical preparation comprising a therapeutically effective amount of (R) -2-methoxy-3- (4'-aminophenyl) propionic acid, or a pharmaceutically acceptable salt thereof. . 9. The pharmaceutical preparation for use according to any one of claims 6-8, wherein the pharmaceutical preparation is for oral administration. 10. The pharmaceutical preparation for use according to any one of claims 6-9, wherein the patient is a human. 11. The pharmaceutical preparation for use according to any one of claims 6-10, wherein the fibrosis is intestinal fibrosis. 12. The pharmaceutical preparation for use according to any one of claims 6-10, wherein the fibrosis is idiopathic pulmonary fibrosis. 13. The pharmaceutical preparation for use in treating a patient suffering from intestinal fibrosis according to any one of claims 6-11, wherein the patient is currently suffering or has suffered from hepatitis B or hepatitis C, or wherein the patient has cirrhosis. 14. The pharmaceutical preparation for use in treating a patient suffering from intestinal fibrosis according to any one of claims 6-11, wherein the patient also suffers from Crohn's disease, inflammatory bowel disease or ulcerative colitis.